It is well recognized that cAMP has important and multiple regulatory roles in the development and function of many different cell types. It is also known that cAMP exerts its effects largely through activation of cAMP- dependent protein kinases (PKAs) and/or the guanine nucleotide exchange protein, Epac. Importantly, the many different processes regulated by cAMP/PKA/Epac are highly controlled by a series of cyclic AMP- degrading phosphodiesterases (PDEs). Moreover, inhibition of individual cyclic nucleotide degrading PDEs by selective and specific inhibitors has been a favorite approach for pharmacologically counteracting a number of pathologies. For example, the drug Viagra(R) works in this manner. However, during the recent grant period we have made the discovery that in at least in two different tissues, inhibition of a single PDE is NOT sufficient to regulate certin physiological processes in that tissue. We have shown, for example, that a classic PDE4 inhibitor is completely ineffective in controlling steroid production in Leydig cells unless the PDE8 activity of these cells is also inhibited. This is a prime example of the process of synergy - several pathway modulators acting together to have a greater effect than either one alone. If this concept turns out to be generally valid in several tissues, it has GREAT implications for the strategies of PDE inhibitor design in the future. Therefore, we plan to investigate whether the same principle holds true in two different tissues that are highly regulated by cAMP and highly express the cAMP-specific PDE8 like steroid producing Leydig cells. We intend to address the question, does more than one PDE need to be inhibited in order to effectively regulate cAMP-dependent processes in these tissues. We will study the effects of multiple PDE inhibitor treatment, including a new PDE8 inhibitor, on thermogenesis in brown adipose tissue (BAT)/brown adipocytes, and on calcium signaling in cardiomyocytes and functional parameters of the heart. Each of these processes is highly cAMP-dependent and each cell type expresses high levels of PDE8A. However, none of them to our knowledge have had their cAMP-dependent processes examined for the effects of PDE8 inhibition alone and particularly not in conjunction with inhibition of other PDEs expressed in these cells. Moreover, we will investigate the molecular mechanisms underlying the synergistic action of multiple PDEs in a variety of cell types including Leydig cells and hepatocytes, for which we have already shown the occurrence of PDE synergies. Over the last 20 years, many drug companies have spent hundreds and hundreds of millions of dollars searching for selective PDE inhibitors to treat various disorders including inflammation, cancer, and obesity. With the exception of erectile dysfunction and pulmonary hypertension (processes regulated by cGMP) these efforts have been only marginally successful. If the proposed studies turnout as expected, they will contribute substantially to explaining why many of these selective inhibitor approaches have shown only minor efficacy. Moreover, the data will likely suggest an alternate approach for the treatment of various diseases with PDE inhibitors by using drugs that target more than one PDE. PUBLIC HEALTH RELEVANCE: The proposed studies will explore the recently discovered synergistic mechanisms by which one of the cAMP-specific phosphodiesterases, PDE8A, works in concert with other cAMP-PDEs to regulate biological processes. The proposed studies will test whether this mechanism of regulation, which we have validated for steroid producing cells, is generally applicable to regulation of other tissues and cell types as well as exploring the molecular mechanisms by which this synergy is mediated.